1. Field of the Invention
The present invention relates generally to microarray spotting instruments and, more particularly, to a method and apparatus for washing and drying pins in such instruments.
2. Description of Related Art
As is well known (and described, e.g., in U.S. Pat. No. 5,807,522 issued to Brown et al. and in “DNA Microarrays: A Practical Approach,” Schena, Mark, N.Y., Oxford University Press, 1999, ISBN 0-19-963776-8), microarrays are arrays of very small samples of purified DNA or protein target material arranged as a grid of hundreds or thousands of small spots on a solid substrate. When the microarray is exposed to selected probe material, the probe material selectively binds to the target spots only where complementary bonding sites occur, through a process called hybridization. Subsequent quantitative scanning in a fluorescent microarray scanner may be used to produce a pixel map of fluorescent intensities (See, e.g., U.S. Pat. No. 5,895,915 issued to DeWeerd et al.). This fluorescent intensity map can then be analyzed by special purpose quantitation algorithms, which reveal the relative concentrations of the fluorescent probes and hence the level of gene expression, protein concentration, etc., present in the cells from which the probe samples were extracted.
Microarray spotting instruments (also known as “spotters”) are used to place the small samples of DNA, protein, or other target biological material onto the microarray substrates. The spotting instruments retrieve the target material from wells in a reservoir plates and “print” target spots in arrays on the microarray substrates. The reservoir plates are typically 96-well or 384-well plates, although other types are also used. Microarrays often have thousands or tens of thousands of target spots, with each spot being of a target compound from a different well of a plate.
FIG. 1 is a simplified block diagram of components of a typical spotter 10. The spotter 10 includes (1) a plate-holding well station 12, which holds one or more reservoir plates, (2) a substrate holding station 14, which holds a plurality of microarray substrates (typically 20-100 substrates), (3) a printhead 16, which holds a plurality of microarray spotting pins (shown, e.g., in FIG. 2), (4) a pin washing and drying apparatus 18, and (5) an actuator system 20, which includes robotic manipulator arms for moving the printhead in X, Y and Z directions relative to the plates, substrates and the washing and drying apparatus. The spotter is enclosed in an enclosure 21 to provide a humidity controlled environment.
FIG. 2 shows a side view of a simplified printhead 16 holding a plurality of pins 22. For convenience of illustration, only two pins 22 are shown, although a typical printhead will hold many more pins. Pins typically include a pinhead 24, a pin shaft 26 and a tapered pin tip 28. The printhead 16 comprises a block of material, typically metal, that includes an array of through-holes. The through-holes are slightly larger than the outer diameter of the pin shafts 26 so the shafts can extend through the through-holes. The through-holes are also smaller than the outer diameter of the pin heads 24 so that when the pin shaft 26 is dropped into one of the through-holes, the pin head 24 will be supported by the upper surface of the printhead 16. The pins are thereby “slip-fit” into the through-holes of the printhead.
Pins are commercially available in several distinct forms. The simplest pins are solid pins. These pins are simple and robust, but in being dipped into the target material in a well typically only take up enough material to form one spot. This then requires the spotting instrument to dip the pin once for every microarray spot that is to be printed.
More commonly used pins are multi-spot dispensing pins that can hold enough target material from a sample reservoir to form multiple spots before they need to be re-dipped in the reservoir. One such type of multi-spot dispensing pin is a slotted pin (shown in FIG. 3), which has a gap or slot 30 at the pin tip 28. One example of such a pin is the MicroQuill brand pin available from Majer Precision Engineering, Inc. This type of pin draws fluid into the gap or slot 30 by capillary action, and deposits a small amount onto the substrate. The amount forming a spot is small compared to the sample uptake volume, so that each dip of the pin into sample liquid takes up enough sample material to print about 50-250 nearly identical spots without re-dipping in the sample plate.
A spotting cycle of a microarray spotting instrument 10 for printing a batch of microarrays is generally as follows: (1) the printhead 16 is moved to the well station 12 and positioned such that the pins 22 are located directly above particular respective wells of a reservoir plate; (2) the printhead 16 is lowered to dip the pins in respective wells, allowing each pin to take up an aliquot of sample; (3) the printhead 16 is moved to the substrate station 14 and positioned above the first substrate to be printed; (4) the printhead 16 is lowered, allowing the tips of the pins to make contact with the substrate, thereby depositing spots of target material on the substrate; (5) the printhead 16 is lifted (so that the pins are no longer in contact with the substrate) and moved to a position above the next substrate to be printed, and the printing motion is repeated; (6) printing is repeated until all substrates in the batch have been printed with these particular samples; (7) the printhead 16 is then moved to a pin washer of the wash/dry apparatus 18, and the pins are washed by dipping them in a wash solution; (8) the printhead 16 is moved to the pin dryer also of the wash/dry apparatus 18, where the pins are dried by inserting them in a vacuum dryer; (9) the wash/dry sequence is repeated two or three times; and (10) the printhead 16 is moved to a position such that the pins 22 are above the next target material to be taken up and the entire cycle is repeated until all desired samples have been printed on the batch of substrates.
As indicated above, microarrays are typically spotted in batches, where the spotter is loaded with a plurality of substrates and the spotting operation produces multiple spotted microarrays, all of which are substantially identically printed. Each microarray typically is spotted so that it has between hundreds and tens of thousands of spots, where each spot comprises the dried residue of a liquid droplet transferred or printed by a pin. The printhead is typically fitted with between 4 and 64 pins, which perform all of their operations in parallel.
After printing the last substrate with a given sample, the pins are washed and dried. This step is important because if the remaining sample is not adequately removed from each pin, the subsequent spots printed by the pin will be contaminated by the “carry-over” from the previous sample. Also, samples in wells subsequently dipped will be contaminated when the pin is dipped for sample uptake. Cross-contamination of samples in a microarray or plate would be very problematic in a microarray application because the hybridization and analysis processes occurring after spotting are based on the assumption that each spot is a single, pure sample and not some mixture of unknown proportions.
Prior art spotters utilize separate pin washing and pin drying devices at the wash/dry station 18. The washing is typically accomplished by dipping the pin tips into a reservoir of wash fluid, typically distilled or filtered water. FIG. 4 illustrates one prior art pin washer 50, in which the wash fluid is kept flowing through a wash chamber 52, in which the pins are dipped. Wash fluid is introduced into a small, open input chamber 54 by a tube fed by a peristaltic pump. The tube is connected to a wash fluid input port 55. The fluid flows over a weir or dam 56 into the slightly larger washing chamber 52. The two-chamber approach allows any sediment in the wash fluid to settle out in the input chamber 54 before the fluid is brought into contact with the pins 22. A wash fluid output or drain port 58 drains fluid from the chamber 52.
Dipping the pin tips in the fluid in the wash chamber 52 causes the pins 22 to take up wash fluid by capillary action in the pin tip slot 30, similar to the way the pins take up sample from the reservoir plates. This dilutes the remaining sample in the pin slot reservoirs.
As shown in FIG. 5, in another prior art wash device 70, an ultrasonic transducer 72 (sometimes called a sonicator) is provided to introduce micro-cavitation in the wash fluid in the wash chamber 74. Sonication is a more effective washing process than the dipping and diluting process of FIG. 4. However, repeated sonication is not recommended because the split tips of pins (defining the slot 30) can act as tuning forks and resonate sympathetically with the ultrasonic signal. This can lead to the pin tip oscillating at high amplitudes and damaging the critical and fragile surfaces near the pin tip.
With the prior art dipping type pin washers, there is a possibility of cross-contamination between pins. The pins of a printhead, which have multiple sample types on them, are dipped simultaneously in the wash fluid, which is a liquid solvent chosen for its ability to dissolve the reagents carried by the pins. The pins are typically only 4.5 to 9.0 mm apart, and some transport of mass from one pin to another during the dip washing process is inevitable even though the likelihood of gross contamination is small.
The pins are then withdrawn from the wash fluid and the printhead is moved to a separate pin drying device. A typical prior art pin dryer 80 is shown in FIG. 6. The pin dryer 80 works by applying vacuum to a plenum chamber 82 below a dryer top plate 84. A vacuum line connects a vacuum pump to the chamber 82 through a port 86. The pin tips are inserted into holes 88 in the dryer top plate 84, and the vacuum below causes ambient air to flow past the pin tips at high velocity and low pressure. These conditions cause the pins, including the slot fluid reservoir in the pin tip, to be dried in about 2-10 seconds. Since the fluid that was dried in and on the pin was diluted by the first washing step, and not entirely washed away, dried residue of diluted sample remains on the pin surface after drying. For this reason, the washing step is repeated at least twice and sometimes as many as four times, with each washing causing a further dilution of the residue in and on the pin until it is inconsequential.
The performance of a vacuum pin dryer 80 depends on the velocity of the air drawn past the pins and on the humidity of that air. Many spotters provide humidity-controlled environments within their enclosures 21, with humidity typically about 55%-65%. Air with that level of humidity is considerably less effective for drying pins than air at the 30%-45% humidity levels most commonly found in office and laboratory environments. The drying time and/or the air velocity must be increased to retain dryer effectiveness at the higher humidity level. Also, vacuum dryers that obtain their air supply from the humidified enclosure 21 generally pump that humidified air out to the atmosphere, placing an additional load on external humidity generating mechanisms and humidity controlling mechanisms.
Prior art dryers typically utilize vacuum pumps of linear piston or rotary vane type, with volume flow ratings in the 1-5 cfm range. When these pumps are applied to dryers with 32 or more pin holes, the air velocity around the pins is generally in the 2-10 m/sec range. Under these conditions, drying times are rarely less than 3 seconds and can be as long as 10 seconds or more. In some instruments, users often block off any dryer holes not being used (as the printhead is often not fully populated with pins) with tape, to increase the velocity of the air in the holes of the dryer that are being used.
The timing of a typical prior art spotting cycle for printing material from one dip into the sample plate onto a batch of 20 microarrays is as shown in the following table:
Number ofTimeoperationsTotal timeOperation(sec.)per cycle(sec.)Move to sample plate position, dip pins,313and withdraw pins.Move to first printing position over a1.211.2substrate, print, and withdraw pins.Move to next substrate, print, and1.21922.8withdraw pins (repeated 19 more times).Move to washer, dip, and withdraw236Move to dryer, insert pins, hold in dryer4312for three seconds, and withdraw pins.Total time45
Of the 45 seconds needed for one printing cycle, 18 seconds or 40% of the time is spent on washing and drying the pins. Accordingly, a quicker washing/drying process would significantly increase the throughput of the spotting instrument.
Many Microarrays have over 10,000 unique spots printed on them. Most printheads are fitted with no more than 8 or 16 pins since, greater numbers of pins cause the footprint of the printed array to be large, which leads to experimental complications at a downstream hybridization step. Printing 10,000 spots with 16 pins requires 625 dip—print—wash/dry cycles in the spotter. Printing 10,000 spots with 8 pins requires 1,250 cycles, of which over six hours (22,500 seconds) is devoted to washing and drying. Thus, the amount of time required to perform the washing and drying can be a significant portion of the total time required to spot a batch of microarrays and also can be a long time period per batch in absolute terms.
A need exists for a faster and more efficient method and apparatus for washing and drying microarray spotting pins in order to improve the throughput of microarray spotting instruments.